Peroxynitrite (ONOO−), an isomer of nitrate, has been known for about one century. During the past decade it has been extensively studied due to its potential important role in biology and medicine (Gryglewski, R., Nature 1986, 320, 454; Beckman, J. S., Am. J. Physiol. Cell Physiol. 1996, 271, C1424; Squadrito, G. L. et al., Free Radical Biol. & Med. 1998, 25, 797; Groves, J. T., Curr. Opin. Chem. Biol. 1999, 3, 226; Radi, R. et al., Free Radical Biol. & Med. 2001, 30, 463-488; Tarpey, M. M. et al., Circ. Res. 2001, 89, 224-236; and Koppenol, W. H., Redox Report 2001, 6, 339-341). Peroxynitrite can be formed in vivo from the diffusion-controlled reaction (k =0.4−1.9×1010 M−1s−1) of nitric oxide (NO) and superoxide (O2.−) in one to one stoichiometry (Hughes, M. N. et al., J. Chem. Soc. (A) 1968, 450) and the concentration of NO is the key controller during the peroxynitrite production process. The reaction between nitric oxide and superoxide proceeds when the concentration of NO increases and can overcome dismutation by superoxide dismutase. This situation occurs when nitric oxide (NO) is overproduced by cytokine-stimulated inducible NO synthase (iNOS). The pathological activity of ONOO− is related to its reaction with the biologically ubiquitous CO2, thereby producing the highly reactive radicals CO3. and NO2. in about 35% yield (Radi, R. et al., Free Radical Biol. & Med. 2001, 30, 463-488). As a result, peroxynitrite can nitrate tyrosine (Ischiropoulos, H., Arch. Biochem. Biophys. 1998, 356, 1-11, and Beckman J. S. et al., Arch Biochem Biophys, 1992, 298, 438-445) and oxidize proteins, lipids (Radi, R. et al., Arch. Biochem. Biophys. 1991, 288, 481, and Shi, H. et al., Biochem. Biophys. Res. Commun. 1999, 257, 651) and iron and sulfur clusters of biological molecules (Radi R, et al., J. Biol. Chem, 1991, 266, 4244-4250). Like other oxidizing agents in living organisms, peroxynitrite and its protonated form have been associated with both beneficial and harmful effects. Macrophages produce peroxynitrite as a host-defense response to bacterial invasion. However, several studies have implicated that peroxynitrite contributes to tissue injury in a number of human diseases such as ischemic reperfusion injury, rheumatoid arthritis, septic shock, multiple sclerosis, atherosclerosis, stroke, inflammatory bowl disease, cancer, and several neurodegenerative diseases (MacMillan-Crow, L. A. et al., Proc. Natl. Acad. Sci. USA 1996, 93, 11853; Rodenas, J. et al., Free Radical. Biol. & Med. 2000, 28, 374; Cuzzocrea, S. et al., Pharmacol Rev. 2001, 53, 135; Szabo, C. Toxicol. Lett. 2003, 140, 105; White, C. R. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 1044; Lipton, S. A. et al., Nature 1993, 364, 626; Pappolla, M. A. et al., J. Neural Transm. 2000, 107, 203; and Beal, M. F., Free Radical Biol. & Med 2002, 32, 392).
Explanation of the critical role of peroxynitrite in living organisms has become increasingly important. Although it is stable in alkaline solution, peroxynitrite decays rapidly upon protonation at physiological pH. The short half-life of peroxynitrite in biological system (1 s in buffers of neutral pH values and less than 100 ms in cells) precludes its direct isolation (Denicola, A. et al. Arch. Biochem. Biophys. 1996, 333, 49-58). Even though solid evidence is known regarding the formation of peroxynitrite in vivo, tools for unambiguous detection and quantitation of peroxynitrite in cells and tissues are not yet available.
Up to now, the available analytical methods for detecting and measuring peroxynitrite can be classified into three types. The first type is the electrochemical sensor, which is used to estimate the amounts of peroxynitrite generated in cells under oxidative stress. (Augusto, O. et al., J. Methods Enzymol. 1996, 269, 346-354; Gatti, R. M. et al., FEBS Lett, 1994, 348, 287-290; Gatti, R. M. et al. Arch. Biochem. Biophys. 1998, 349, 36-46; and Karoui, H. et al., J. Biol. Chem. 1996, 271, 6000-6009). But this method requires manipulation of sophisticated apparatus and does not allow spatial imaging of peroxynitrite.
The second type relies on the employment of oxidation probes. For example, DCFH (2′,7′-dichlordihydrofluorescein) and DHR 123 (dihydrorhodamine 123), which can be oxidized by peroxynitrite to yield highly fluorescent molecules, have been used for monitoring peroxynitrite in cells and tissues (Royall, J. A. et al., Arch. Biochem. Biophys. 1993, 302, 348-355; Kooy, N. W. et al., Free Radic. Biol. Med. 1994, 16, 149-156; Kooy, N. W. et al., Free Radic. Biol. Res. 1997,27,245-254; Crow, J. P. Nitric Oxide. 1997, 1, 145-157; Ischiropoulos, H et al., Methods Enzymol. 1999, 301, 367-373; and Miles, A. M. et al., J. Biol. Chem. 1996, 271, 40-47). However, the mechanism of oxidation of DCFH and DHR by peroxynitrite remains largely unknown and these probes can also be oxidized by many other ROS (reactive oxygen species) produced by cells. A similar problem can be found in luminal chemiluminescence system for detecting peroxynitrite in cell culture solution. HPF (hydroxyphenyl fluorescein) can distinguish between peroxynitrite and nitric oxide, but it gives out higher fluorescent signal with hydroxyl radical than does with peroxynitrite (Setsukinai, K. et al., J. Biol. Chem. 2003, 278, 3170-3175; International Publication No. WO 01/64664 (Nagano et al.); and International Publication No. WO2004040296 (Nagano et al.)).
The third type utilizes the footprinting reaction of biological molecules. For example, 3-nitrotyrosine, a nitration product generated after oxidation of tyrosine residues of proteins by peroxynitrite in biological systems, can be detected by immunochemical methods (Kaur, H.; et al., FEBS Lett. 1994, 350, 9-12). NADH (reduced nicotamide adenine dinucleotide) has also been used recently for monitoring of peroxynitrite concentration in buffers by fluorescence. However, at the moment, there are no entirely specific chemical modifications of either probes or biomolecules that can directly indicate the generation of perokynitrite in cells in an unambiguous manner. It implies that other reactive oxygen species and reactive nitrogen species present in the biological systems may compete with peroxynitrite and interfere with the results.
Several methods have been known for the detection/measurement of peroxynitrite, including electrochemical method, the chemiluminescence method and the footprinting method. However, these methods require tedious and time-consuming control experiments using a combination of scavengers and inhibitors, and give low sensitivity and specificity. Thus, to facilitate the direct studies of peroxynitrite in biological system, it is of ultimate importance to develop methods specific for peroxynitrite detection and measurement which are highly sensitive and easy to operate.